U.S. patent application number 13/499876 was filed with the patent office on 2012-09-27 for pharmaceutical composition containing medicament-containing fine particles and method for producing same.
This patent application is currently assigned to NITTO DENKO CORPORATION. Invention is credited to Martyn C. Davies, Lisbeth Illum, Chieko Kitaura, Kenjiro Minomi, Toshihiko Okazaki, Katsuyuki Okubo, Elizabeth Pearson, Clive J. Roberts, Snjezana Stolnik-Trenkic.
Application Number | 20120244196 13/499876 |
Document ID | / |
Family ID | 43826402 |
Filed Date | 2012-09-27 |
United States Patent
Application |
20120244196 |
Kind Code |
A1 |
Okubo; Katsuyuki ; et
al. |
September 27, 2012 |
PHARMACEUTICAL COMPOSITION CONTAINING MEDICAMENT-CONTAINING FINE
PARTICLES AND METHOD FOR PRODUCING SAME
Abstract
The invention provides a pharmaceutical composition that can be
used for an efficient administration of a water-soluble polymer
drug by a method other than injection, and a method for production
of the pharmaceutical composition. The pharmaceutical composition
contains a small particle comprised of (a) a water-soluble drug and
(b) a pharmaceutically acceptable ionic crystalline compound which
is solid at room temperature, wherein the ionic crystalline
compound is crystallized in the small particle.
Inventors: |
Okubo; Katsuyuki; (Osaka,
JP) ; Okazaki; Toshihiko; (Osaka, JP) ;
Kitaura; Chieko; (Osaka, JP) ; Minomi; Kenjiro;
(Osaka, JP) ; Pearson; Elizabeth; (Basel, CH)
; Roberts; Clive J.; (Nottingham, GB) ; Davies;
Martyn C.; (Nottingham, GB) ; Stolnik-Trenkic;
Snjezana; (Nottingham, GB) ; Illum; Lisbeth;
(Nottingham, GB) |
Assignee: |
NITTO DENKO CORPORATION
Ibaraki-shi, Osaka
JP
|
Family ID: |
43826402 |
Appl. No.: |
13/499876 |
Filed: |
October 1, 2010 |
PCT Filed: |
October 1, 2010 |
PCT NO: |
PCT/JP2010/067241 |
371 Date: |
June 5, 2012 |
Current U.S.
Class: |
424/400 ;
514/15.2; 514/5.9 |
Current CPC
Class: |
A61K 38/28 20130101;
A61K 38/28 20130101; A61K 33/30 20130101; A61K 39/385 20130101;
A61K 9/146 20130101; A61K 9/19 20130101; A61K 48/00 20130101; A61K
33/14 20130101; C12N 15/88 20130101; A61K 31/715 20130101; A61K
33/30 20130101; A61K 33/14 20130101; A61P 3/10 20180101; A61K
31/713 20130101; A61K 31/711 20130101; A61K 33/00 20130101; A61K
2039/55555 20130101; A61K 33/00 20130101; A61K 39/39 20130101; A61K
45/06 20130101; A61K 31/7105 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/400 ;
514/5.9; 514/15.2 |
International
Class: |
A61K 9/14 20060101
A61K009/14; A61K 38/38 20060101 A61K038/38; A61K 38/28 20060101
A61K038/28 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 2, 2009 |
JP |
2009-231001 |
Claims
1. A pharmaceutical composition comprising a small particle
comprised of (a) a water-soluble drug and (b) a pharmaceutically
acceptable ionic crystalline compound which is solid at room
temperature, wherein the ionic crystalline compound is crystallized
in the small particle.
2. The pharmaceutical composition of claim 1, wherein the small
particle is free of irreversible crosslinking.
3. The pharmaceutical composition of claim 1, wherein the small
particle has an average particle size of not less than 10 nm and
not more than 5000 nm.
4. The pharmaceutical composition of claim 1, wherein the
water-soluble drug is selected from the group consisting of
peptide, protein, DNA, RNA, siRNA, polysaccharide, lipopeptide,
lipoprotein, lipopolysaccharide, low-molecular-weight compound,
antibody, antigen, toxin and vaccine.
5. The pharmaceutical composition of claim 1, wherein the ionic
crystalline compound comprises as constituent components, one or
more types of cationic electrolytes selected from the group
consisting of sodium ion, potassium ion, calcium ion, magnesium
ion, zinc ion, iron ion and ammonium ion, and one or more types of
anionic electrolytes selected from the group consisting of chlorine
ion, sulfuric acid ion, lactic acid ion, acetic acid ion,
phosphoric acid ion, gluconic acid ion, carbonate ion and
bicarbonate ion.
6. The pharmaceutical composition of claim 1, wherein the
water-soluble drug has a weight ratio of not less than 0.001 and
not more than 100 relative to the ionic crystalline compound.
7. The pharmaceutical composition of claim 1, further comprising
(c) a pharmaceutically acceptable polymer, wherein the small
particle has a positive or negative charge at a predetermined pH,
and the polymer has a charge of a sign opposite to that of the
small particle at said pH, whereby the small particle and the
polymer electrostatically interact with each other at said pH to
form a complex wherein the polymer is attached to the surface of
the small particle.
8. The pharmaceutical composition of claim 7, wherein the complex
has an average particle size of not less than 15 nm and not more
than 6000 nm.
9. A production method of the pharmaceutical composition of claim
1, which comprises drying without heating an aqueous solution of
the water-soluble drug and the ionic crystalline compound under
reduced pressure.
10. The method of claim 9, wherein the drying is freeze-drying or
drying by centrifugal concentration.
11. A method of producing the pharmaceutical composition of claim
7, comprising drying without heating an aqueous solution of the
water-soluble drug and the ionic crystalline compound under reduced
pressure to give the small particle and mixing the solution of the
polymer with said pH and the small particle.
12. The method of claim 11, wherein the drying is freeze-drying or
drying by centrifugal concentration.
13. The pharmaceutical composition of claim 2, wherein the small
particle has an average particle size of not less than 10 nm and
not more than 5000 nm.
14. The pharmaceutical composition of claim 13, wherein the
water-soluble drug is selected from the group consisting of
peptide, protein, DNA, RNA, siRNA, polysaccharide, lipopeptide,
lipoprotein, lipopolysaccharide, low-molecular-weight compound,
antibody, antigen, toxin and vaccine.
15. The pharmaceutical composition of claim 14, wherein the ionic
crystalline compound comprises as constituent components, one or
more types of cationic electrolytes selected from the group
consisting of sodium ion, potassium ion, calcium ion, magnesium
ion, zinc ion, iron ion and ammonium ion, and one or more types of
anionic electrolytes selected from the group consisting of chlorine
ion, sulfuric acid ion, lactic acid ion, acetic acid ion,
phosphoric acid ion, gluconic acid ion, carbonate ion and
bicarbonate ion.
16. The pharmaceutical composition of claim 15, wherein the
water-soluble drug has a weight ratio of not less than 0.001 and
not more than 100 relative to the ionic crystalline compound.
17. The pharmaceutical composition of claim 16, further comprising
(c) a pharmaceutically acceptable polymer, wherein the small
particle has a positive or negative charge at a predetermined pH,
and the polymer has a charge of a sign opposite to that of the
small particle at said pH, whereby the small particle and the
polymer electrostatically interact with each other at said pH to
form a complex wherein the polymer is attached to the surface of
the small particle.
18. The pharmaceutical composition of claim 17, wherein the complex
has an average particle size of not less than 15 nm and not more
than 6000 nm.
Description
TECHNICAL FIELD
[0001] The present invention relates to a pharmaceutical
composition comprising a small particle comprised of a
water-soluble drug and an ionic crystalline compound and a
production method thereof.
BACKGROUND ART
[0002] The advances of biotechnology have resulted in the discovery
of a large number of therapeutic compounds such as peptides,
proteins, polysaccharides, polynucleic acids, siRNAs, RNAs,
antibodies, antigens and the like. The physicochemical
characteristics of these compounds (e.g., large molecular weight,
hydrophilicity, instability) make them difficult to administer into
the body by other means than by injection. For some of these drugs
a multiple daily dosing by injection is necessary and results in
non-compliance especially among younger patients (non-patent
documents 1 and 2).
[0003] When administered via an oral route or a transmucosal route
such as pulmonary, transoral (oral mucosal), transvaginal and
transnasal routes, and the like, these drugs are not easily
absorbed across the mucosal surfaces due to their physical size and
hydrophilicity. Furthermore, these drugs are prone to degradation
by the enzymes such as peptidases and proteases, which is
especially a problem in the gastrointestinal tract. In order to
improve the transport of these drugs across mucosal surfaces,
formulations containing absorption enhancers have been used with
some success especially in delivery by the transnasal route and by
the pulmonary route. However, there is a demand for the development
of effective methods and compositions to provide the transport of
high molecular weight compounds across mucosal surfaces.
[0004] Nanoparticles encapsulating drugs have been described as a
mean of providing such transport (non-patent document 3). However,
the encapsulation of peptides and proteins into nanoparticles is
difficult due to the large size of these compounds and the normally
hydrophobic environment in the matrix of a nanoparticle and results
generally in a very low loading capacity and hence the need for
large quantities of nanoparticles to be administered to the mucosal
surface. This has to some extent been solved by the production of
nanocrystals where the major part of the nanoparticle is pure drug.
However, nanocrystal suspensions are generally very unstable and
crystal growth normally takes place in a stepwise manner unless the
crystal is stabilized. The normal procedure for stabilizing
nanocrystals has been through a crosslinking procedure that to some
extent crosslinks the drug itself hence lowering the bioactivity of
the drug or rendering drugs such as proteins immunogenic.
Furthermore, it is evident from publications in the literature that
transport of nanoparticles across the mucosa is not readily
achievable (non-patent document 3).
[0005] Nanoparticles have been widely investigated as carriers for
drug delivery across the mucosa (non-patent documents 3-5).
Nanoparticles have especially been of importance for peptides and
proteins as outlined above. However, problems have been described
in terms of lack of efficiency of the nanoparticle system to
deliver sufficient quantities of drug across the mucosa, low
drug-loading efficiency and compromised stability of the peptide
and protein drugs if not encapsulated into the matrix of the
nanoparticle. Nanoparticles in the form of nanocrystals of a
peptide or a protein have been described in the literature as drug
delivery systems for transmucosal and parenteral delivery
(non-patent documents 6-7). However, the nanocrystals described in
the literature are often physically unstable or if crosslinked to
increase stability will lose some of the biological activity.
Furthermore, often the nanocrystals have a size larger than 1 .mu.m
and hence are microcrystals rather than nanocrystals.
DOCUMENT LIST
Patent Documents
[0006] patent document 1: US2004/02192224 [0007] patent document 2:
WO98/46732 [0008] patent document 3: U.S. Pat. No. 7,087,246 [0009]
patent document 4: WO02/072636 [0010] patent document 5:
WO99/55310
Non-Patent Documents
[0010] [0011] non-patent document 1: Drug Discovery Today. 7;
1184-1189 (2002) [0012] non-patent document 2: J. Control. Rel. 87;
187-198 (2003) [0013] non-patent document 3: J. Pharm. Sci. 96;
473-483 (2007) [0014] non-patent document 4: Biomaterials 23;
3193-3201 (2002) [0015] non-patent document 5: Int J Pharm. 342;
240-249 (2007) [0016] non-patent document 6: J. Biotech. 113;
151-170 (2004) [0017] non-patent document 7: Pharm. Res. 22(9);
1461-1470 (2005)
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[0018] As mentioned above, encapsulation of a water-soluble polymer
drug such as peptide, protein and the like in a small particle with
high efficiency, and preparation of a small particle containing a
water-soluble polymer drug in an appropriate size is not easy. In
addition, it is not easy, when resuspending a composition
containing a small dry particle in water when in use, to avoid
aggregation and afford a redispersion of a particle having a small
size.
[0019] It is an object of the present invention to provide a
pharmaceutical composition that can be used for an efficient
administration of a water-soluble drug by methods other than
injection. More specifically, it is an object of the present
invention to provide a small particle-containing pharmaceutical
composition to be used for efficient transmucosal or transdermal
administration of a water-soluble drug (e.g., peptide, protein,
DNA, RNA, siRNA, polysaccharide, lipopeptide, lipoprotein,
lipopolysaccharide, low-molecular-weight compound, antibody,
antigen, toxin, vaccine and the like), particularly, a small
particle-containing pharmaceutical composition which enables
encapsulation of a water-soluble drug in a small particle with high
efficiency and preparation of a small particle in an appropriate
size, as compared to a conventional small particle-containing
pharmaceutical composition. It is also an object of the present
invention to provide a method for production of the pharmaceutical
composition.
Means of Solving the Problems
[0020] The present inventors focused on transmucosal and
transdermal administration using a small particle system such as
nanoparticle as a method for efficiently administering the
above-mentioned water-soluble drug by a method other than
injection, and conducted diligent investigations. As a result, they
have found that a small particle containing a water-soluble drug
and an ionic crystalline compound, wherein the ionic crystalline
compound is crystallized in the small particle, enables
encapsulation of a water-soluble drug in a small particle with high
efficiency and preparation of a small particle in an appropriate
size, as compared to a conventional small particle-containing
pharmaceutical composition. The present inventors have further
found that coating of the surface of a small particle with a
suitable polymer by electrostatic interaction enables stabilization
of small particles in a suspension. Based on these findings, the
present inventors found that a drug delivery system superior to
conventional methods can be achieved by using a pharmaceutical
composition containing the small particle, which resulted in the
completion of the present invention.
[0021] Accordingly, the present invention is as follows.
[1] A pharmaceutical composition comprising a small particle
comprised of (a) a water-soluble drug and (b) a pharmaceutically
acceptable ionic crystalline compound which is solid at room
temperature, wherein the ionic crystalline compound is crystallized
in the small particle. [2] The pharmaceutical composition of the
above-mentioned [1], wherein the small particle is free of
irreversible crosslinking. [3] The pharmaceutical composition of
the above-mentioned [1] or [2], wherein the small particle has an
average particle size of not less than 10 nm and not more than 5000
nm. [4] The pharmaceutical composition of any one of the
above-mentioned [1] to [3], wherein the water-soluble drug is
selected from the group consisting of peptide, protein, DNA, RNA,
siRNA, polysaccharide, lipopeptide, lipoprotein,
lipopolysaccharide, low-molecular-weight compound, antibody,
antigen, toxin and vaccine. [5] The pharmaceutical composition of
any one of the above-mentioned [1] to [4], wherein the ionic
crystalline compound comprises as constituent components, one or
more types of cationic electrolytes selected from the group
consisting of sodium ion, potassium ion, calcium ion, magnesium
ion, zinc ion, iron ion and ammonium ion, and one or more types of
anionic electrolytes selected from the group consisting of chlorine
ion, sulfuric acid ion, lactic acid ion, acetic acid ion,
phosphoric acid ion, gluconic acid ion, carbonate ion and
bicarbonate ion. [6] The pharmaceutical composition of any one of
the above-mentioned [1] to [5], wherein the water-soluble drug has
a weight ratio of not less than 0.001 and not more than 100
relative to the ionic crystalline compound. [7] The pharmaceutical
composition of any one of the above-mentioned [1] to [6], further
comprising (c) a pharmaceutically acceptable polymer, wherein the
small particle has a positive or negative charge at a predetermined
pH, and the polymer has a charge of a sign opposite to that of the
small particle at said pH, whereby the small particle and the
polymer electrostatically interact with each other at said pH to
form a complex wherein the polymer is attached to the surface of
the small particle. [8] The pharmaceutical composition of the
above-mentioned [7], wherein the complex has an average particle
size of not less than 15 nm and not more than 6000 nm. [9] A
production method of the pharmaceutical composition of any one of
the above-mentioned [1] to [6], which comprises drying without
heating an aqueous solution of the water-soluble drug and the ionic
crystalline compound under reduced pressure. [10] The method of the
above-mentioned [9], wherein the drying is freeze-drying or drying
by centrifugal concentration. [11] A method of producing the
pharmaceutical composition of the above-mentioned [7] or [8],
comprising drying without heating an aqueous solution of the
water-soluble drug and the ionic crystalline compound under reduced
pressure to give the small particle and mixing the solution of the
polymer with said pH and the small particle. [12] The method of the
above-mentioned [11], wherein the drying is freeze-drying or drying
by centrifugal concentration.
Effect of the Invention
[0022] The pharmaceutical composition of the present invention
enables efficient transmucosal or transdermal administration of a
water-soluble drug (e.g., peptide, protein, DNA, RNA, siRNA,
polysaccharide, lipopeptide, lipoprotein, lipopolysaccharide,
low-molecular compound, antibody, antigen, toxin and vaccine and
the like) to a mammal, since a small particle contained in the
pharmaceutical composition has an appropriate particle size and can
concentrate the water-soluble drug in the small particle.
[0023] Moreover, in the pharmaceutical composition of the present
invention comprising a complex wherein the surface of a small
particle is coated with a polymer, the complex has high stability
(e.g., preservation stability, stability against enzymes, and the
like), and permits adjustment of a drug release rate according to
the preparation conditions and the kind of the polymer. Therefore,
the release property desirable for sustained-release preparation
and vaccine can be realized.
[0024] According to the production method of the present invention,
moreover, a water-soluble drug (e.g., peptide, protein, DNA, RNA,
siRNA, polysaccharide, lipopeptide, lipoprotein,
lipopolysaccharide, low-molecular-weight compound, antibody,
antigen, toxin and vaccine and the like) can be encapsulated in a
small particle in a high yield. Moreover, according to the
production method of the present invention, a dried small particle
free of moisture by freeze-drying and the like can be obtained
easily, whereby the preservation stability of a water-soluble drug
can be ensured.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 shows the results of the particle size measurement in
Example 1, wherein the horizontal axis shows the particle size
(nm), the vertical axis shows the scattering intensity distribution
(index value), and the particle size is shown by the particle
diameter.
[0026] FIG. 2 shows the results of the particle size measurement in
Example 2, wherein the horizontal axis shows the particle size
(nm), the vertical axis shows the scattering intensity distribution
(index value), and the particle size is shown by the particle
diameter.
[0027] FIG. 3 shows the results of the particle size measurement in
Comparative Example 1, wherein the horizontal axis shows the
particle size (nm), the vertical axis shows the scattering
intensity distribution (index value), and the particle size is
shown by the particle radius.
[0028] FIG. 4 shows the results of the particle size measurement in
Example 3, wherein the horizontal axis shows the particle size
(nm), the vertical axis shows the scattering intensity distribution
(index value), and the particle size is shown by the particle
radius.
[0029] FIG. 5 shows the results of the particle size measurement in
Comparative Example 2, wherein the horizontal axis shows the
particle size (nm), the vertical axis shows the scattering
intensity distribution (index value), and the particle size is
shown by the particle diameter.
[0030] FIG. 6 shows the results of the particle size measurement in
Example 4, wherein the horizontal axis shows the particle size
(nm), the vertical axis shows the scattering intensity distribution
(index value), and the particle size is shown by the particle
diameter.
[0031] FIG. 7 shows the results of the particle size measurement in
Example 5, wherein the horizontal axis shows the particle size
(nm), the vertical axis shows the scattering intensity distribution
(index value), and the particle size is shown by the particle
diameter.
[0032] FIG. 8 shows the results of the particle size measurement in
Example 6, wherein the horizontal axis shows the particle size
(nm), the vertical axis shows the scattering intensity distribution
(index value), and the particle size is shown by the particle
radius.
[0033] FIG. 9 shows the results of the particle size measurement in
Example 7, wherein the horizontal axis shows the particle size
(nm), the vertical axis shows the scattering intensity distribution
(index value), and the particle size is shown by the particle
radius.
[0034] FIG. 10 shows the results of the particle size measurement
in Example 8, wherein the horizontal axis shows the particle size
(nm), the vertical axis shows the scattering intensity distribution
(index value), and the particle size is shown by the particle
radius.
[0035] FIG. 11 shows the results of the particle size measurement
in Example 9, wherein the horizontal axis shows the particle size
(nm), the vertical axis shows the scattering intensity distribution
(index value), and the particle size is shown by the particle
diameter.
[0036] FIG. 12 shows the results of the particle size measurement
in Example 10, wherein the horizontal axis shows the particle size
(nm), the vertical axis shows the scattering intensity distribution
(index value), and the particle size is shown by the particle
diameter.
[0037] FIG. 13 shows the results of the particle size measurement
in Example 11, wherein the horizontal axis shows the particle size
(nm), the vertical axis shows the scattering intensity distribution
(index value), and the particle size is shown by the particle
diameter.
[0038] FIG. 14 shows the results of the particle size measurement
in Comparative Example 3, wherein the horizontal axis shows the
particle size (nm), the vertical axis shows the scattering
intensity distribution (index value), and the particle size is
shown by the particle radius.
[0039] FIG. 15 shows the results of the particle size measurement
in Comparative Example 4, wherein the horizontal axis shows the
particle size (nm), the vertical axis shows the scattering
intensity distribution (index value), and the particle size is
shown by the particle radius.
[0040] FIG. 16 shows a TEM image of the small particle of Example
10.
[0041] FIG. 17 shows the results of the elemental analysis of the
small particles in the TEM image of Example 10.
[0042] FIG. 18 shows a TEM image of the small particle of Example
11.
[0043] FIG. 19 shows the results of the elemental analysis of the
small particles in the TEM image of Example 11.
[0044] FIG. 20 shows the results of the stability against enzyme
test of chitosan-coated insulin small particle in Experimental
Example 4, wherein the horizontal axis shows the percentage of the
insulin content after shaking reaction at 37.degree. C. for 30 min
relative to the initial content, from top to bottom in the order of
i) a mixed sample of a surface-coated small particle and an enzyme,
ii) a mixed sample of a surface-coated small particle and a buffer,
iii) a mixed sample of an insulin solution and an enzyme, and iv) a
mixed sample of an insulin solution and a buffer.
EMBODIMENT OF THE INVENTION
[0045] The present invention provides a pharmaceutical composition
comprising a small particle comprised of (a) a water-soluble drug
and (b) a pharmaceutically acceptable ionic crystalline compound
which is solid at room temperature, wherein the ionic crystalline
compound is crystallized in the small particle.
[0046] The pharmaceutical composition can be used for, for example,
transmucosal or transdermal administration. Accordingly, the
pharmaceutical composition of the present invention can be
administered to the mucosa (e.g., mucosa such as that in the nose,
mouth cavity, eye, vagina and gastrointestinal tract) or skin of a
subject in need of treatment and/or therapy by an appropriate
method such as coating, spraying, nebulizing, patching and the
like, whereby a drug can be delivered to the mucosa or skin tissue,
or circulatory or immunity system via mucosa or skin tissue to
provide efficacy, vaccine effect and the like.
[0047] The above-mentioned "water-soluble drug" is appropriately
selected according to the intended use of a pharmaceutical
composition. The drug may be crystalline or noncrystalline. In the
present specification, "water-soluble" means compatibility with
water at ambient temperature. The choice of the drug is not
particularly limited as long as the drug shows a minimal solubility
in water at ambient temperature. More specifically, while the
water-soluble drug usable for a pharmaceutical composition of the
present invention is not limited to the following, for example, it
is preferably dissolved in water (100 g) at 25.degree. C. in an
amount of not less than 0.0001 g, more preferably not less than
0.001 g, still more preferably not less than 0.01 g. As a solvent
in which the drug is dissolved, a mixture of an organic solvent
(ethanol and the like) and water, an aqueous acid solution and
aqueous alkali solution can be used, in addition to simple water,
and an appropriate solvent can be appropriately selected according
to the chemical property of the drug. As the drug, a drug that can
be dissolved in an organic solvent that can be mixed with water,
such as methanol, ethanol, isopropanol, acetone, acetonitrile and
the like, or a drug that can be dissolved in a mixture of such
organic solvent and water is preferable.
[0048] The molecular weight of the drug is not particularly
limited, and may be a low-molecular-weight compound or a polymer
compound. The low-molecular-weight compound in the present
specification means a compound having a molecular weight of less
than 500 D, and the polymer compound in the present specification
means a compound having a molecular weight of not less than 500 D.
Examples of the drug include peptide, protein, DNA, RNA, siRNA,
polysaccharide, lipopeptide, lipoprotein, lipopolysaccharide,
low-molecular-weight compound, antibody, antigen, toxin and vaccine
and the like. Examples of the drug also include various peptides,
proteins, DNAs, RNAs, siRNAs, polysaccharides, lipopeptides,
lipoproteins, lipopolysaccharides, low-molecular-weight compounds,
antibodies, antigens, toxins, vaccines and the like such as
antihypertensive agent, antihypotensive agent, analgesic,
antipsychotic agent, antidepressant, antimanic, antianxiety agent,
sedative, hypnotic, antiepileptic, opioid agonist, therapeutic
agent for asthma, anesthetic, antiarrhythmic agent, therapeutic
agent for arthritis, antiepileptic drugs, ACE inhibitor,
decongestant, antibiotic, antianginal agent, diuretic,
antiparkinson agent, bronchodilator, oxytocic, antidiuretic,
antilipemic agent, immunosuppressant, immunity regulator,
antiemetic, antiinfective agent, antineoplastic, antifungal agent,
antivirus agent, antidiabetic agent, antiallergic agent, fever
reducer, antitumor agent, antigout agent, antihistamine agent,
antipruritic agent, bone regulator, cardiovascular agent,
hypocholesterolemic agent, antimalarial agent, pharmaceutical agent
for ceasing smoking, antitussive agent, expectorant, mucolytic
agent, nasal decongestant, dopamine agonist, pharmaceutical agent
for digestive tract, muscle relaxant, neuromuscular blocker,
parasympatholytic, prostaglandin, stimulant drug, anorectic agent,
thyroid agent or antithyroid agent, hormone, antimigraine agent,
antiobestic agent, antiinflammatory agent and the like.
[0049] Specific examples of the drug include, but are not limited
to, insulin, glucagon, leuprolide, growth hormones, parathyroid
hormones, calcitonin, vascular endothelial growth factor,
erythropoietin, heparin, cyclosporin, oxytocin, tyrosine,
enkephalin, tyrotropin releasing hormone, follicle-stimulating
hormone, leuteinising hormone, vasopressin, vasopressin analogs,
catalase, superoxide dismutase, interleukin II, interferons, colony
stimulating factor, tumor necrosis factor, melanocyte stimulating
hormone, glucagon-like peptide-1, glucagon-like peptide-2,
katacalcin, cholecystekinin-12, cholecystekinin-8, exendin,
gonadoliberin-related peptide, insulin-like protein,
leucine-enkephalin, methionine-enkephalin, leumorphine,
neurophysin, copeptin, neuropeptide Y, neuropeptide AF,
PACAP-related peptide, pancreatic hormone, peptide YY, urotensin,
intestinal peptide, adrenocorticotropic peptide, epidermal growth
factor, prolactin, luteinising hormone releasing hormone (LHRH),
LHRH agonist, growth hormone releasing factor, somatostatin,
gastrin, tetragastrin, pentagastrin, endorphins, angiotensins,
tyrotropin releasing hormone, granulocyte-colony stimulating
factor, granulocyte-macrophage-colony stimulating factor,
heparinase, antigens for influenza vaccine, tetanus toxins,
diphtheria toxin, cancer antigen protein, cancer antigen peptide,
.beta.-amyloid, immunoglobulins, siRNAs for treatment of cirrhosis,
siRNAs for treatment of cancer, low-molecular compounds such as
bromhexine, granisetron, zolmitriptan, sumatriptan and the like,
and pharmaceutically acceptable salts thereof, and the like. It is
also possible to appropriately use two or more kinds of drugs in
combination.
[0050] The choice of the above-mentioned "pharmaceutically
acceptable ionic crystalline compound which is solid at room
temperature" is not particularly limited as long as it is an ionic
crystalline compound which is solid at room temperature and
pharmaceutically acceptable. A compound, having a melting point,
decomposition temperature, or sublimation temperature of not less
than 40.degree. C. is preferable, not less than 100.degree. C. is
more preferable, and not less than 200.degree. C. is still more
preferable. A compound having a melting point, decomposition
temperature, or sublimation temperature of less than 40.degree. C.
is not preferable since it cannot provide a stable solid crystal in
a small particle, and shows low ability to precipitate or form a
particle of a water-soluble drug. While the upper limit of the
melting point, decomposition temperature, or sublimation
temperature of the compound is not particularly limited, the
melting point, decomposition temperature, or sublimation
temperature is preferably not more than 2000.degree. C., more
preferably not more than 1800.degree. C., still more preferably not
more than 1500.degree. C.
[0051] As mentioned above, the ionic crystalline compound is
crystallized in small particles. The "crystallization" here is not
particularly limited as long as crystals of the ionized crystalline
compound are dispersed inside the small particle comprised of the
water soluble compound and the ionic crystalline compound when the
small particle is observed by, for example, a preferable apparatus
such as a transmission electron microscope and the like. As shown
in the below-mentioned Examples, whether the crystal is derived
from the compound can be confirmed by elemental analysis of
crystals by, for example, XMA. It is considered that a salt is
locally contained at high concentration by crystallization of ionic
crystalline compound in small particles, which enables efficient
precipitation of a water-soluble drug in the area surrounding the
salt and particle formation, as well as inhibits aggregation of the
drug to prevent small particles from having a greater size.
[0052] The ionic crystalline compound usable for the pharmaceutical
composition of the present invention includes, but is not limited
to, for example, an ionic crystalline compound comprising, as
constituent components, one or more kinds of cationic electrolytes
selected from the group consisting of sodium ion, potassium ion,
calcium ion, magnesium ion, zinc ion, iron ion and ammonium ion,
and one or more kinds of anionic electrolyte selected from the
group consisting of chlorine ion, sulfuric acid ion, lactic acid
ion, acetic acid ion, phosphoric acid ion, gluconic acid ion,
carbonate ion and bicarbonate ion. Among those, examples of
preferable ionic crystalline compound include sodium chloride,
potassium chloride, ammonium sulfate and sodium sulfate, since they
are highly safe to the body and show good reproducibility of drug
precipitation due to high regularity of crystal shape of the
salt.
[0053] While the weight ratio of the water-soluble drug to the
ionic crystalline compound in the pharmaceutical composition of the
present invention is not particularly limited as long as a small
particle with preferable characteristics that meets a desired
object can be obtained, it is typically not less than 0.001 and not
more than 100, preferably not less than 0.005 and not more than 50,
more preferably not less than 0.01 and not more than 10. By
selecting such weight ratios, efficient drug precipitation and
formation of salt crystal can be realized.
[0054] The water content of the small particle varies depending on
the state of the surrounding area. For example, it is negligible in
a dry state, but water is considered to penetrate somewhat into the
small particle in water. Since the water content of the small
particle in water is essentially irrelevant to the structure and
property of the small particle, the range of the water content of
the small particle is also not particularly limited. For example,
it is 0.01-99.99 wt %.
[0055] Moreover, the small particle is preferably free of
irreversible crosslinking such as covalent bonds, from the aspects
of physiological activity, immunogenicity and the like, as
mentioned above. The irreversible crosslinking here does not
include pseudo crosslinking (e.g., electrostatic interaction) that
can be reversibly dissociated by addition of water.
[0056] The size of the small particle in the pharmaceutical
composition of the present invention is preferably an average
particle size of 10-5000 nm, more preferably 20-4000 nm, still more
preferably 50-3000 nm. A size of the small particle of greater than
5000 nm is not preferable, since even when the same amount of a
drug is administered, the number of small particles decreases, the
total surface area of the small particles reduces and the drug
release rate becomes low. It is also undesirable since the
efficiency of uptake by immunocompetent cells decreases when used
as a particle for vaccine. In addition, a size of the small
particle of smaller than 10 nm is not preferable, since the amount
of a drug contained per one small particle becomes too small, and
the pharmacological effect or vaccine effect after administration
becomes weak. It is also undesirable since small particles easily
aggregate during production or use, and the handling becomes
difficult.
[0057] The particle size here refers to a value obtained by
measuring small particles dispersed in water, which is a diameter
calculated on the assumption that the small particles have a
spherical shape. To be specific, the particle size is measured by a
dynamic light scattering measuring apparatus, and an average of the
hydrodynamic diameter determined from the scattering intensity
distribution is employed as an average particle size. Here, an
average particle size of small particles of, for example, not less
than 10 nm means that not less than 10%, preferably not less than
20%, more preferably not less than 30%, still more preferably not
less than 40%, particularly preferably not less than 50% of the
average particle size of peaks is not less than 10 nm in the
proportion of each particle size peak in the above-mentioned
scattering intensity distribution by the dynamic light scattering
measuring apparatus (proportion of cumulative scattering intensity
for each particle size peak to cumulative scattering intensity for
all peaks).
[0058] The pharmaceutical composition of the present invention
optionally further contains a pharmaceutically acceptable polymer.
In this case, it is possible that the small particles are
constituted to have a positive or negative charge at a
predetermined pH, and a polymer having a charge of the sign
opposite to that of the small particle at said pH is used as the
polymer, whereby they are allowed to electrostatically interact
with each other to form a complex having the polymer attached to
the surface of the small particle.
[0059] In other words, the present invention also provides a
pharmaceutical composition comprising a small particle comprised of
(a) a water-soluble drug and (b) a pharmaceutically acceptable
ionic crystalline compound which is solid at room temperature,
wherein the ionic crystalline compound is crystallized in the small
particle,
[0060] said pharmaceutical composition further comprises (c) a
pharmaceutically acceptable polymer, and
[0061] the small particle that has a positive or negative charge at
a predetermined pH, and the polymer has a charge of a sign opposite
to that of the small particle at said pH, whereby the small
particle and the polymer electrostatically interact with each other
at said pH to form a complex wherein the polymer is attached to the
surface of the small particle.
[0062] As long as the small particle has a positive or negative
charge at a predetermined pH, the aforementioned water-soluble
drug, ionic crystalline compound and small particle can also be
used for this embodiment.
[0063] In the following, when a polymer is attached to the surface
of the small particle, it is sometimes expressed that a small
particle is "coated" with the polymer and the like, and a complex
wherein a polymer is attached to the surface of the small particle,
it is sometimes referred to as a "surface-coated small particle".
Furthermore, a small particle free of coating with a polymer is
sometimes referred to as a "small core particle" or simply as a
"core particle", so as to expressly distinguish a surface-coated
small particle. Coating of a small core particle with a polymer is
not particularly limited as long as a polymer is attached to the
surface of a small core particle by an electrostatic interaction
between the small particle and the polymer.
[0064] The pharmaceutically acceptable polymer making up the
surface-coated small particle (hereinafter to be referred to as a
"surface-coating polymer") is preferably biocompatible. The term
"biocompatibility" herein means that a substance and decomposed
products thereof have no toxic or hazardous effect on a body tissue
or a body system (e.g., blood circulation system, nerve system,
immune system and the like) and cause no undue immune rejection.
The biocompatible polymer is suitable for administration to human
or other animals. In addition, the polymer is more preferably
biodegradable. The term "biodegradability" herein means that a
substance is decomposed within a living body by an enzymatic,
chemical or physical process or the like within an acceptable
period of time to form a smaller chemical species. Methods for
examining biocompatibility and biodegradability of a substance are
well known in the technical field of the invention.
[0065] The surface-coating polymer may be a natural polymer or a
synthetic polymer. Since the surface-coating polymer
electrostatically interacts with the small particle on the surface
of the small particle at the predetermined pH, it needs to be a
polymer having a charge of the sign opposite to that of the small
particle at the pH. Where necessary, more than one polymer can be
used in combination for the surface-coating polymers as long as
they are capable of having charges of the same sign at the pH.
[0066] The pharmaceutical composition of the present invention
comprising the surface-coated small particle can be produced, for
example, by the below-mentioned production method; when the
composition is produced by the production method, even if the
surface-coating polymer is by itself slightly water-soluble at the
predetermined pH, the composition can be produced by mixing the
surface-coating polymer with the small particle at a pH where the
surface-coating polymer is readily water-soluble, and then
adjusting the pH of the mixture to the predetermined pH. Therefore,
as the surface-coating polymer, not only polymers that are readily
water-soluble at the predetermined pH, but also polymers that are
slightly water-soluble at the predetermined pH can be used.
[0067] As the polymer that can be used for the surface-coating
polymer, polyanionic or polycationic polysaccharides, polyamino
acids and other charged polymers can be mentioned without limit.
The polymer is appropriately selected based on the kind of the drug
used, the charge, and the like.
[0068] Polyanionic polysaccharides that can be used in the present
invention means a polysaccharide that has one or more acidic polar
groups such as a carboxyl group, a sulfuric acid group or a
phosphoric acid group in the constitutional unit. Examples of such
polyanionic polysaccharides include, but are not limited to,
chondroitin sulfuric acid, dextran sulfuric acid,
carboxymethylcellulose, alginic acid, pectin, hyaluronic acid,
derivatives and salts thereof and the like.
[0069] Polycationic polysaccharides that can be used in the present
invention means a polysaccharide that has one or more basic polar
group such as an amino group in the constitutional unit. Examples
of such polycationic polysaccharides include, but are not limited
to, chitin, chitosan, derivatives and salts thereof and the like.
Chitosan and chitosan derivative can be selected from those having
various molecular weights and degrees of deacetylation, and
chitosan derivative can be selected from those having various
degrees of substitution.
[0070] The polyanionic polyamino acid that can be used in the
present invention means a polyamino acid whose isoelectric point is
on the acidic side of the physiological pH; examples thereof
include, but are not limited to, polyglutamic acid, polyaspartic
acid, derivatives and salts thereof and the like.
[0071] The polycationic polyamino acid that can be used in the
present invention means a polyamino acid whose isoelectric point is
on the basic side of the physiological pH; examples thereof
include, but are not limited to, polylysine, polyarginine,
derivatives and salts thereof and the like.
[0072] Examples of the polymer that can be used for the
surface-coating polymer other than the above-mentioned
polysaccharides and polyamino acids, include polyethylenimine,
polyacryl acid, derivatives and salts thereof and the like.
[0073] The surface-coating polymer may be polyethylene glycolated
(PEGylated) and/or glycosylated.
[0074] The surface-coating polymer may be mucoadhesive, and/or act
as a transmucosal absorption promoter. Examples of mucoadhesive
polymers include chitosan, polyacryl acid, sodium alginate,
carboxymethylcellulose and the like as well as PEGylated polymers
thereof and the like. Examples of the polymers that functions as
transmucosal absorption promoters include chitosan, polyacryl acid,
polyarginine, salts and derivatives thereof and the like.
[0075] Those ordinarily skilled in the art can determine the
molecular weight of a surface-coating polymer in consideration of
factors such as degradation rate, mechanical strength, solubility,
which kind of small particle would form the complex with the
surface-coating polymer, and the like. Typically, the weight
average molecular weight of the surface-coating polymer should
preferably be not less than 1,000 Da, and more preferably not less
than 2,000 Da; preferably not more than 1,000,000 Da, and more
preferably not more than 500,000 Da, as measured by gel permeation
chromatography. Accordingly, typically, the weight average
molecular weight of the surface-coating polymer is preferably
between 1,000-1,000,000 Da, and more preferably between
2,000-500,000 Da. For example, the weight average molecular weight
of chitin or chitosan may be between 1,000-1,000,000 Da. and the
degree of deacetylation of chitin or chitosan may be between
20-100%.
[0076] The size of the surface-coated small particle is, for
example, an average particle size of preferably 15-6000 nm, more
preferably 25-5000 nm, still more preferably 55-4000 nm. When the
surface-coated small particle size is greater than 6000 nm, the
efficiency of uptake by immunocompetent cells unpreferably
decreases when used as a small particle for vaccine. A
surface-coated small particle size smaller than 15 nm is not
preferable, since small particles easily aggregate during
production or use, and the handling becomes difficult.
[0077] The particle size here refers to a value obtained by
measuring surface-coated small particles dispersed in water, and a
diameter calculated on the assumption that the surface-coated small
particles have a spherical shape. Specifically, the particle size
is measured by a dynamic light scattering measuring apparatus, and
an average of the hydrodynamic diameter determined from the
scattering intensity distribution is employed as an average
particle size. Here, an average particle size of surface-coated
small particles of, for example, not less than 15 nm means that not
less than 10%, preferably not less than 20%, more preferably not
less than 30%, still more preferably not less than 40%,
particularly preferably not less than 50% of the average particle
size of peaks is not less than 15 nm in the proportion of each
particle size peak in the above-mentioned scattering intensity
distribution by the dynamic light scattering measuring apparatus
(proportion of cumulative scattering intensity for each particle
size peak to cumulative scattering intensity for all peaks).
[0078] As the particle size of small core particle in
surface-coated small particles, a particle size observed by a
microscope such as TEM, SEM, AFM and the like may be employed.
[0079] The preferable relative proportions of the drug, the ionic
crystalline compound and the surface-coating polymer in the
pharmaceutical composition of the present invention comprising the
surface-coated small particle vary depending on the kind of the
drug, ionic crystalline compound and surface-coating polymer to be
used, the size of the small core particle and the like and hence
cannot be stated in general. The relative proportion as expressed
by the weight ratio in a pharmaceutical composition may be, for
example, drug:ionic crystalline compound:surface-coating
polymer=1:0.01-1000:0.1-1000.
[0080] The predetermined pH is desirably set to a suitable
physiological pH of the administration site so as to avoid topical
irritation that occurs when the pharmaceutical composition of the
present invention is administered to the body. As mentioned above,
the pharmaceutical composition of the present invention can be
administered to mucosa such as that in the lung, mouth cavity, eye,
vagina, intestine, nose and the like or skin, where the
physiological pH varies in these various mucosas or skin. For
example, the physiological pH of the gastrointestinal tract
increases along the length thereof from about pH 1 in the stomach
to pH 8 in the colon; the mouth cavity has a pH around 6.8; the pH
of nasal fluid is within the range of about pH 5.5 to 6.5; the pH
of vagina is around 4.5. For example, when the pharmaceutical
composition of the present invention is to be administered to the
nasal mucosa, preferable pH value of the composition is, for
example, about 6.0.
[0081] The pharmaceutical composition of the present invention may
be incorporated into any dosage form as long as the small particle
(i.e., small core particle or surface-coated small particle) can
directly reach the target site. Examples thereof include pulmonary
agent, oral agent, buccal agent, intraocular agent, vaginal agent,
intranasal agent, suppository, percutaneous absorber and the
like.
[0082] As the pulmonary agent, an inhalant which is delivered to
alveoli by some lung inhaler is preferred.
[0083] As the oral agent, usual oral preparations, for example,
tablet, granule, fine granule, capsule and the like can be
mentioned. Dosage forms designed to release the drug in the small
intestine, for example, enteric tablet, enteric granule, enteric
capsule and enteric fine granule are preferred.
[0084] As the buccal agent, the intraocular agent and the
intranasal agent, buccal tablet, buccal spray, eye drop, nasal
drop, aerosol, ointment, gel, cream, liquid, suspension, lotion,
dry powder, sheet, patch and the like can be mentioned.
[0085] As the vaginal agent and suppository, ointment, gel, cream,
liquid, suspension, lotion, dry powder, sheet, capsule and the like
can be mentioned.
[0086] The transdermal absorbent is not particularly limited and
may be in any dosage form as long as the drug can be absorbed from
the skin. For example, ointment, gel, cream, gel cream, liquid,
lotion, aerosol, liniment, plaster, adhesive skin patch, reservoir
patch and the like can be mentioned. These dosage forms may be used
alone and noninvasively, of in a combination with an invasive
device such as microneedle, stratum corneum peeling tape, ablation
and the like.
[0087] As a method for preparing the above-mentioned dosage forms,
known production methods generally used in the field can be
applied. Where necessary, when preparing the above-mentioned dosage
forms, carriers such as excipient, binder, disintegrant and
lubricant, and various preparation additives such as sweetening
agent, surfactant, suspending agent, emulsifier, colorant,
preservative and stabilizer, which are generally used for preparing
the particular dosage forms, can be appropriately added in an
appropriate quantity to produce the dosage forms. Also, the
pharmaceutical composition of the present invention can be
preserved in the form of a dry powder prepared by lyophilizing the
suspension, and the like, and resuspended by adding water to the
dry powder when in use. Employing this method, hydrolysis can be
avoided to improve the preservation stability of the pharmaceutical
composition.
[0088] The ratio of the small particle in the pharmaceutical
composition of the present invention (i.e., small core particle or
surface-coated small particle) is preferably 0.01-100 wt %, more
preferably 0.1-100 wt %.
[0089] The pharmaceutical composition of the present invention is
stable and with low toxicity, and can be used safely. The
administration frequency and single dose vary depending on the drug
used, condition and body weight of patient, administration route,
therapeutic strategy and the like and hence cannot be stated in
general. For example, when the composition of the present invention
in which insulin is used as the drug, is transnasally administered
to a patient with diabetes and the like, as one therapeutic
strategy, about 2 to about 6 mg of the active ingredient (insulin)
can be administered to an adult (about 60 kg in body weight) before
each meal.
[0090] The present invention also provides a production method of
the aforementioned pharmaceutical composition. It should be noted
that the pharmaceutical composition of the present invention can
also be produced by a production method other than the method
explained below.
[0091] The above-mentioned small core particle can be prepared by a
method comprising drying an aqueous solution of the above-mentioned
water-soluble drug and the above-mentioned ionic crystalline
compound under reduced pressure without heating.
[0092] The concentration of a drug in an aqueous solution
containing a drug and an ionic crystalline compound is preferably
0.01-30 wt %, more preferably 0.05-20 wt %. When the drug
concentration is lower than 0.01 wt %, the composition ratio of the
drug in a pharmaceutical composition to be produced becomes
unpreferably small, and the production yield becomes unpreferably
low. A drug concentration higher than 30 wt % is not preferable,
since the particle size of particles obtained by drying cannot be
made small.
[0093] The concentration of an ionic crystalline compound in an
aqueous solution containing a drug and an ionic crystalline
compound is preferably 0.1-50 wt %, more preferably 0.5-30 wt %. A
concentration of the ionic crystalline compound of lower than 0.1
wt % is not preferable, since drug precipitation and particle
formation cannot be efficiently performed. A concentration of the
ionic crystalline compound of higher than 50 wt % is not
preferable, since drug precipitation starts before drying and small
core particles having a small size cannot be produced.
[0094] As mentioned above, in this method, drying is performed
under reduced pressure without heating. The "drying" here means an
operation to remove water from the above-mentioned aqueous solution
substantially completely or partially.
[0095] Drying under reduced pressure is performed under a pressure
of preferably 0-10000 Pa, more preferably 0-600 Pa, still more
preferably 0-200 Pa. A pressure higher than 10000 Pa is not
preferable since water cannot be removed efficiently.
[0096] In this method, the drying without heating is performed
under a temperature condition of preferably -200.degree. C. to
40.degree. C., more preferably -100.degree. C. to 35.degree. C.
Drying at a temperature of higher than 40.degree. C. is not
preferable since the drug solubility is improved, and the speed of
drug precipitation and particle formation by drying operation
becomes slow. As a result, the particle size becomes large.
[0097] For the above-mentioned drying, freeze-drying or centrifugal
concentration drying can be utilized.
[0098] The freeze-drying in this production method comprises
rapidly cooling to freeze the above-mentioned aqueous solution of a
drug and an ionic crystalline compound using a freezer or liquid
nitrogen, promptly setting the frozen product on a freeze-dryer and
removing without heating water under reduced pressure. By this
operation, the above-mentioned small core particle can be prepared.
As a freeze-dryer, any types of dryers used in the art can be used
appropriately. For example, freeze dryer DC400 of Yamato Scientific
Co., Ltd. can be mentioned. In a production method using
freeze-drying, the above-mentioned aqueous solution is frozen by
rapidly cooling using a freezer or liquid nitrogen, whereby the
drug can be rapidly insolubilized and precipitated. As a result, a
small core particle having a smaller particle size can be
preferably produced. It is also preferable since scaled-up
production is possible by utilizing freeze-drying.
[0099] In the drying by centrifugal concentration in the production
method, the above-mentioned aqueous solution of a drug and an ionic
crystalline compound is treated by centrifugation using a suitable
centrifugal concentration dryer and dried without heating under
reduced pressure to remove water. By this operation, the
above-mentioned small core particles can be prepared. As the
centrifugal concentration dryer, any type used in the art can be
appropriately used. For example, Jouan concentrator evaporator
system (RC10.22) and Concentrator 5301 manufactured by Eppendorf
can be mentioned. Since the production method using centrifugal
concentration drying permits drug precipitation and particle
formation by removing water at ambient temperature, it can be
utilized as an alternative means to avoid freeze-drying when the
stability of the drug is influenced by a freeze-drying operation
and the like.
[0100] The above-mentioned surface-coated small particles can be
prepared by a method including mixing small core particles obtained
as mentioned above with a solution of a surface-coating polymer
having the above-mentioned predetermined pH. When the
surface-coating polymer is poorly soluble at said pH, a
surface-coated small particle can be prepared by a method including
dissolving the surface-coating polymer in a solution at pH at which
the surface-coating polymer is readily soluble, adding the small
core particle, and adjusting the pH of the mixture to the
predetermined pH. By these methods, a suspension of the
surface-coated small particles can be obtained. The obtained
suspension may or may not be dialyzed.
[0101] A suspension of small core particles or surface-coated small
particles obtained by the aforementioned method is formulated into
the above-mentioned suitable dosage form by a known production
method generally used in the technical field. As mentioned above,
where necessary, when the above-mentioned dosage form is prepared,
a carrier such as excipient, binder, disintegrant and lubricant,
and various preparation additives such as sweetening agents,
surfactants, suspending agents, emulsifiers, colorants,
preservatives and stabilizers, which are generally used for
preparing the particular dosage form can be appropriately added in
an appropriate quantity to produce the dosage forms. Also, the
pharmaceutical composition of the present invention can be produced
in the form of a dry powder prepared by lyophilizing the
suspension, and the like.
[0102] While the present invention is hereinafter further explained
in detail by referring to Examples, Comparative Examples and
Experimental Examples, the present invention is not limited by the
following Examples and the like.
EXAMPLES
[0103] In the following Examples and Comparative Examples, the
particle size of particles in a suspension was measured by a
dynamic light scattering method using DLS 802 (Viscotek) or
ELS-8000 (Otsuka Electronics Co., Ltd.). The zeta potential of the
particle of each sample was measured using Zeta sizer nano
(Malvern).
Example 1
Preparation of Uncoated Insulin Core Particle by Centrifugal
Concentration Drying
[0104] In this Example, insulin (isoelectric point (pI)=5.3) was
used as a protein drug, and insulin core particles (uncoated) were
prepared by centrifugal concentration to remove water from an
insulin solution containing an ionic crystalline compound to allow
precipitation of insulin.
[0105] The preparation included the following steps.
[0106] Hydrochloric acid (0.02 M) (3 ml) was added to dissolve
Bovine Insulin (Sigma) (30 mg), and 2 ml of a salt solution
(trisodium citrate dihydrate 58 mg/ml, zinc chloride 1.4 mg/ml,
sodium chloride 180 mg/ml) was added. Thereafter, NaOH (0.1 M) was
added to adjust the pH to 6.25. The obtained solution (0.25 ml) was
transferred to a glass vial, and water was removed without heating
by centrifugal concentrator under reduced pressure to dry the
solution. Water (2 ml) was added to the dried product to give a
suspension for particle size measurement.
[0107] The size of the particles in the obtained suspension was
measured. As a result, the diameter of the particles in the main
resultant product was 730 nm (FIG. 1). Accordingly, it was found
that core particles having a small particle size can be obtained by
the above-mentioned preparation method.
[0108] In addition, the zeta potential of the particles in the
obtained suspension was measured. As a result, the zeta potential
was -10 mV at pH 6. Reflecting the minus charge of insulin at pH 6,
the core particles were also charged minus.
Example 2
Preparation of Uncoated Insulin Core Particle by Freeze-Drying
[0109] In this Example, insulin (pI=5.3) was used as a protein
drug, and insulin core particles (uncoated) were prepared by
freeze-drying to remove water from an insulin solution containing
an ionic crystalline compound to allow precipitation of
insulin.
[0110] The preparation included the following steps.
[0111] Hydrochloric acid (0.02 M) (3 ml) was added to dissolve
Bovine Insulin (Sigma) (30 mg), and 2 ml of a salt solution
(trisodium citrate dihydrate 58 mg/ml, zinc chloride 1.4 mg/ml,
sodium chloride 180 mg/ml) was added. Thereafter, NaOH (0.1 M) was
added to adjust the pH to 6.25. The obtained solution (0.25 ml) was
transferred to a glass vial, frozen with liquid nitrogen and water
was removed without heating by a freeze drier under reduced
pressure to dry the solution. Water (2 ml) was added to the dried
product to give a suspension for particle size measurement.
[0112] The size of the particles in the obtained suspension was
measured. As a result, the diameter of the particles in the main
resultant product was 180 nm (FIG. 2). Accordingly, it was found
that core particles having a small particle size can also be
obtained by the above-mentioned preparation method.
Comparative Example 1
Preparation of Uncoated Insulin Core Particles by Cooling
[0113] In this Comparative Example, insulin (pI=5.3) was used as a
protein drug, and preparation of insulin core particles (uncoated)
was tried by cooling an insulin solution containing an ionic
crystalline compound to allow precipitation of insulin.
[0114] The preparation included the following steps.
[0115] Hydrochloric acid (0.02 M) (3 ml) was added to dissolve
Bovine Insulin (Sigma) (30 mg), and 2 ml of sodium citrate (0.2 M),
24 .mu.l of zinc chloride solution (0.12 g/ml), and 0.36 g of
sodium chloride were added and mixed therewith. Thereafter, a small
amount of NaOH (0.1 M) was added to adjust the pH to 6.25. The
obtained solution (1 ml) was transferred to a glass vial and cooled
overnight at 4.degree. C. to allow crystal precipitation, whereby
an insulin particle suspension was obtained.
[0116] The size of the particles in the obtained suspension was
measured. As a result, the particles diameter varied greatly from 6
nm considered to be a gathering of several molecules of insulin to
a large aggregate, showing insufficient control of the particle
size (FIG. 3).
Example 3
Preparation of Uncoated Insulin Core Particles by Centrifugal
Concentration Drying and Cooling
[0117] In this Example, insulin (pI=5.3) was used as a protein
drug, and insulin core particles (uncoated) were prepared by
removing a part of water from an insulin solution containing an
ionic crystalline compound by centrifugal concentration to allow
precipitation of insulin, and promoting precipitation by
cooling.
[0118] The preparation included the following steps.
[0119] Hydrochloric acid (0.02 M) (3 ml) was added to dissolve
Bovine Insulin (Sigma) (30 mg), and 2 ml of sodium citrate (0.2 M),
24 .mu.l of zinc chloride solution (0.12 g/ml), and 0.36 g of
sodium chloride were added and mixed therewith. Thereafter, a small
amount of NaOH (0.1 M) was added to adjust the pH to 6.25. The
obtained solution (1 ml) was transferred to a glass vial, and dried
without heating by centrifugal concentrator under reduced pressure
for 15 min to remove a part of water. Then, the solution was cooled
at 4.degree. C. overnight to continue crystal precipitation,
whereby an insulin particle suspension was obtained.
[0120] The size of the particles in the obtained suspension was
measured. As a result, the diameter of the particles in the main
resultant product was 390 nm (FIG. 4). Therefore, it was confirmed
that insulin core particles having good and uniform particle size
can be obtained by drying without heating by centrifugal
concentrator under reduced pressure, even without complete
drying.
Comparative Example 2
Preparation of Insulin Core Particles by Centrifugal Concentration
Drying of Insulin Solution Free of Ionic Crystalline Compound
[0121] In this Comparative Example, insulin (pI=5.3) was used as a
protein drug, and insulin core particles (uncoated) were prepared
by removing water from an insulin solution free of an ionic
crystalline compound by centrifugal concentration drying to allow
precipitation of insulin.
[0122] The preparation included the following steps.
[0123] Hydrochloric acid (0.02 M) (3 ml) was added to dissolve
Bovine Insulin (Sigma) (30 mg), and 2 ml of additive solution
(trisodium citrate dihydrate 58 mg/ml, zinc chloride 1.4 mg/ml) was
added. Thereafter, NaOH (0.1 M) was added to adjust the pH to 6.25.
The obtained solution (0.25 ml) was transferred to a glass vial,
and dried without heating by centrifugal concentrator under reduced
pressure to remove water. Water (2 ml) was added to the dried
product to give a suspension for particle size measurement.
[0124] The size of the particles in the obtained suspension was
measured. As a result, the diameter of the particles in the main
resultant product was 3200 nm (FIG. 5). When the surface of core
particles is coated, the size grows more than that. Therefore, a
particle size of this level is not suitable for use desired in the
present invention, particularly, use for vaccine, and is not
preferable.
[0125] From the results of Examples 1-3 and Comparative Examples
1-2, it is clear that addition of an ionic crystalline compound to
an insulin solution, and insulin precipitation by drying without
heating under reduced pressure are effective for preparation of
insulin core particles (uncoated) having an appropriate particle
size. It is considered that insulin precipitation and preparation
of ionic crystalline compound simultaneously proceed by drying
without heating under reduced pressure, thereby preventing
excessive aggregation and of large size of the precipitated insulin
particles.
Example 4
Preparation of Surface-Coated Small Particle by Coating Insulin
Core Particle Prepared by Centrifugal Concentration Drying with
Chitosan
[0126] In this Example, surface-coated small particles were
prepared by coating insulin core particles prepared by centrifugal
concentration drying with chitosan.
[0127] The preparation included the following steps.
[0128] Hydrochloric acid (0.02 M) (3 ml) was added to dissolve
Bovine Insulin (Sigma) (30 mg), and 2 ml of salt solution
(trisodium citrate dihydrate 58 mg/ml, zinc chloride 1.4 mg/ml,
sodium chloride 180 mg/ml) was added. Thereafter, NaOH (0.1 M) was
added to adjust the pH to 6.25. The obtained solution (0.25 ml) was
transferred to a glass vial, and dried without heating by
centrifugal concentrator under reduced pressure to remove water. To
the dried product was added 1 ml of chitosan solution (manufactured
by Dainichiseika Co., Ltd., aqueous chitosan solution) prepared to
1% (w/v) to give a resuspension, which was placed in a dialysis
tube (molecular weight cutoff value 10000) and dialyzed against 500
ml of 5 mM MES buffer (pH 6.0) for 60 min. A suspension after
dialysis was obtained as a sample.
[0129] The size of the particles in the obtained suspension was
measured. As a result, the diameter of the particles in the main
resultant product was 1800 nm (FIG. 6). As compared to the insulin
core particles of Example 1 prepared by a similar method, the
particle size increased.
[0130] In addition, the zeta potential of the particles in the
obtained suspension was measured. As a result, the zeta potential
was +19 mV at pH 6. The insulin core particles of Example 1 were
charged minus reflecting the minus charge of insulin at pH 6. The
small particles obtained in this Example had a plus charge derived
from the plus charge of chitosan at pH 6.
[0131] Therefrom it is clear that insulin core particles were
coated fine with chitosan.
Example 5
Preparation of Surface-Coated Small Particles by Coating Insulin
Core Particles Prepared by Freeze-Drying with Chitosan
[0132] In this Example, insulin core particles prepared by
freeze-drying were coated with chitosan, whereby surface-coated
small particles were prepared.
[0133] The preparation included the following steps.
[0134] Hydrochloric acid (0.02 M) (3 ml) was added to dissolve
Bovine Insulin (Sigma) (30 mg), and 2 ml of salt solution
(trisodium citrate dihydrate 58 mg/ml, zinc chloride 1.4 mg/ml,
sodium chloride 180 mg/ml) was added. Thereafter, NaOH (0.1 M) was
added to adjust the pH to 6.25. The obtained solution (0.25 ml) was
transferred to a glass vial, frozen with liquid nitrogen, and dried
without heating by freeze-drier under reduced pressure to remove
water. To the dried product was added 1 ml of chitosan solution
(manufactured by Dainichiseika Co., Ltd., aqueous chitosan
solution) prepared to 1% (w/v) to give a resuspension, which was
placed in a dialysis tube (molecular weight cutoff value 10000) and
dialyzed against 500 ml of 5 mM MES buffer (pH 6.0) for 60 min. A
suspension after dialysis was obtained as a sample.
[0135] The size of the particles in the obtained suspension was
measured. As a result, the diameter of the particles in the main
resultant product was 1500 nm (FIG. 7). As compared to the insulin
core particles of Example 2 prepared by a similar method, the
particle size increased.
[0136] In addition, the zeta potential of the particles in the
obtained suspension was measured. As a result, the zeta potential
was +17 mV at pH 6.
[0137] Therefrom it is clear that surface-coated small particles
having a small particle size can also be obtained by a method using
freeze-drying as in Example 4.
Example 6
Preparation of Surface-Coated Small Particles by Coating Insulin
Core Particles with DMAEMA-PEG (without Dialysis)
[0138] In this Example, insulin core particles prepared by
centrifugal concentration drying were coated with DMAEMA-PEG,
whereby surface-coated small particles were prepared. For
preparation, dialysis was not performed.
[0139] The preparation included the following steps.
[0140] Hydrochloric acid (0.02 M) (3 ml) was added to dissolve
Bovine Insulin (Sigma) (30 mg), and 2 ml of trisodium citrate (0.2
M), 24 .mu.l of zinc chloride solution (0.12 g/ml), and 0.36 g of
sodium chloride were added. Thereafter, a small amount of NaOH (0.1
M) was added to adjust the pH to 6.25. The obtained solution (1 ml)
was transferred to a glass vial, and dried without heating by
centrifugal concentrator under reduced pressure to completely
remove water. To the dried product was added 1 ml of 0.1% w/v
aqueous DMAEMA-stat-PEGMA solution (pH 6.0) to give a resuspension
as a sample.
[0141] DMAEMA-stat-PEGMA is a compound having the following
chemical formula:
##STR00001##
[0142] The size of the particles in the obtained suspension was
measured. As a result, the diameter of the particles in the main
resultant product was 260 nm (FIG. 8).
Example 7
Preparation of Surface-Coated Small Particles by Coating Insulin
Core Particles with DMAEMA-PEG (with Dialysis)
[0143] A suspension prepared in the same manner as in Example 6 was
placed in a dialysis tube (molecular weight cutoff value 10000) and
dialyzed against 500 ml of 0.5 mM citric acid solution (pH 6.0) for
60 min. A suspension after dialysis was obtained as a sample.
[0144] The size of the particles in the obtained suspension was
measured. As a result, the diameter of the particles in the main
resultant product was 260 nm (FIG. 9).
[0145] In Examples 6 and 7, DMAEMA-PEG was used as a
surface-coating polymer. In this case, it was confirmed that stable
surface-coated small particles having a small particle size can be
prepared irrespective of use of dialysis.
Example 8
Preparation of Surface-Coated Small Particles by Coating Insulin
Core Particles with PEG-Modified Chitosan
[0146] In this Example, insulin core particles prepared by
centrifugal concentration drying were coated with PEG-modified
chitosan, whereby surface-coated small particles were prepared.
[0147] The preparation included the following steps.
[0148] Hydrochloric acid (0.02 M) (3 ml) was added to dissolve
Bovine Insulin (Sigma) (30 mg), and 2 ml of trisodium citrate (0.2
M), 24 .mu.l of zinc chloride solution (0.12 g/ml), and 0.36 g of
sodium chloride were added. Thereafter, a small amount of NaOH (0.1
M) was added to adjust the pH to 6.25. The obtained solution (1 ml)
was transferred to a glass vial, and dried without heating by
centrifugal concentrator under reduced pressure to completely
remove water. To the dried product was added 1 ml of 0.1% w/v
aqueous solution (pH 6.0) of PEG-modified chitosan (manufactured by
Carbomer.Inc.) to give a resuspension as a sample.
[0149] The size of the particles in the obtained suspension was
measured. As a result, the diameter of the particles in the main
resultant product was 510 nm (FIG. 10).
[0150] It was confirmed that stable surface-coated small particles
having a small particle size can also be prepared when PEG-modified
chitosan was used as a surface-coating polymer.
Example 9
Preparation of Surface-Coated Small Particles by Coating Egg
Albumin Core Particles with Chitosan
[0151] In this Example, surface-coated small particles were
prepared by coating egg albumin core particles prepared by
centrifugal concentration drying with chitosan.
[0152] The preparation included the following steps.
[0153] Hydrochloric acid (0.02 M) (2.4 ml) was added to dissolve
egg albumin (Sigma) (24 mg), and 1.6 ml of salt solution (trisodium
citrate dihydrate 59 mg/ml, sodium chloride 180 mg/ml) was added.
Thereafter, NaOH (0.1 M) was added to adjust the pH to 6.25. The
obtained solution (0.25 ml) was transferred to a glass vial, and
dried without heating by centrifugal concentrator under reduced
pressure to remove water. To the dried product was added 1 ml of
1.5% (w/v) chitosan solution (manufactured by Dainichiseika Co.,
Ltd., aqueous chitosan solution) to give a resuspension, which was
placed in a dialysis tube (molecular weight cutoff value 10000) and
dialyzed against 500 ml of 5 mM MES buffer (pH 6.0) for 60 min. A
suspension after dialysis was obtained as a sample.
[0154] The size of the particles in the obtained suspension was
measured. As a result, the diameter of the particles in the main
resultant product was 600 nm (FIG. 11).
[0155] In addition, the zeta potential of the particles in the
obtained suspension was measured. As a result, the zeta potential
was +12 mV at pH 6.
[0156] In this Example, coated particles were prepared using egg
albumin as a protein drug and by centrifugal concentration drying
excluding zinc chloride. It was confirmed that surface-coated small
particles having a small particle size can be prepared in this
case.
Example 10
Preparation of Surface-Coated Small Particles by Coating Egg
Albumin Core Particles with Cationic Chitosan Derivative (without
Dialysis)
[0157] In this Example, surface-coated small particles were
prepared by coating egg albumin core particles prepared by
freeze-drying with chitosan. For preparation, dialysis was not
performed.
[0158] The preparation included the following steps.
[0159] Hydrochloric acid (0.02 M) (2.4 ml) was added to dissolve
egg albumin (Sigma) (24 mg), and 1.6 ml of salt solution (trisodium
citrate dihydrate 59 mg/ml, sodium chloride 180 mg/ml) was added.
Thereafter, NaOH (0.1 M) was added to adjust the pH to 6.25. The
obtained solution (0.25 ml) was transferred to a glass vial, frozen
with liquid nitrogen, and dried without heating by freeze-drier
under reduced pressure to remove water. To the dried product was
resuspended in 1 ml of 1.1% (w/v) cationic aqueous chitosan
derivative solution (manufactured by Dainichiseika Co., Ltd.,
cationic chitosan derivative) to give a surface-coated small
particles. Then, the particles were diluted with 5 mM MES buffer
(pH 6.5) to give a sample having an egg albumin concentration of
62.5 .mu.g/ml.
[0160] The size of the particles in the obtained suspension was
measured. As a result, the diameter of the particles in the main
resultant product was 260 nm (FIG. 12).
[0161] In addition, the zeta potential of the particles in the
obtained suspension was measured. As a result, the zeta potential
was +21 mV at pH 6.5.
Example 11
Preparation of Surface-Coated Small Particles by Coating Egg
Albumin Core Particles with Cationic Chitosan Derivative (with
Dialysis)
[0162] In this Example, egg albumin core particles prepared by
freeze-drying were coated with chitosan to give surface-coated
small particles. For preparation, dialysis was performed.
[0163] The preparation included the following steps.
[0164] Hydrochloric acid (0.02 M) (2.4 ml) was added to dissolve
egg albumin (Sigma) (24 mg), and 1.6 ml of salt solution (trisodium
citrate dihydrate 59 mg/ml, sodium chloride 180 mg/ml) was added.
Thereafter, NaOH (0.1 M) was added to adjust the pH to 6.25. The
obtained solution (0.25 ml) was transferred to a glass vial, frozen
with liquid nitrogen, and dried without heating by freeze-drier
under reduced pressure to remove water. To the dried product was
added 1 ml of 1.1% (w/v) cationic chitosan solution (manufactured
by Dainichiseika Co., Ltd., cationic chitosan derivative) to give a
resuspension, which was placed in a dialysis tube (molecular weight
cutoff value 10000) and dialyzed twice each against 500 ml of 5 mM
MES buffer (pH 6.5) for 30 min. A suspension after dialysis was
obtained as a sample.
[0165] The size of the particles in the obtained suspension was
measured. As a result, the diameter of the particles in the main
resultant product was 484 nm (FIG. 13).
[0166] In addition, the zeta potential of the particles in the
obtained suspension was measured. As a result, the zeta potential
was +21 mV at pH 6.5.
[0167] In Examples 10 and 11, egg albumin was used as a protein
drug and a cationic chitosan derivative was used as a
surface-coating polymer. In these cases, it was confirmed that
stable surface-coated small particles having a small particle size
can be prepared irrespective of use of dialysis.
Comparative Example 3
Preparation of Small Particles Using Tween 80 Instead of
Polymer
[0168] In this Comparative Example, preparation of surface-coated
small particles was tried in the same manner as in Example 6 except
that Tween 80 was used instead of aqueous DMAEMA-stat-PEGMA
solution.
[0169] The preparation included the following steps.
[0170] Hydrochloric acid (0.02 M) (3 ml) was added to dissolve
Bovine Insulin (Sigma) (30 mg), and 2 ml of trisodium citrate (0.2
M), 24 .mu.l of zinc chloride solution (0.12 g/ml), and 0.36 g of
sodium chloride were added. Thereafter, a small amount of NaOH (0.1
M) was added to adjust the pH to 6.25. The obtained solution (1 ml)
was transferred to a glass vial, and dried without heating by
centrifugal concentrator under reduced pressure to completely
remove water. To the dried product was added 1 ml of 0.1% w/v
aqueous solution of Tween 80 (pH 6.0) to give a resuspension as a
sample.
[0171] The particle size of the particles in the obtained
suspension was measured. As a result, the diameter of the particles
in the main resultant product was over 5000 nm (FIG. 14).
Comparative Example 4
Preparation of Small Particles at pH Different from Example 8
[0172] In this Example, small particles were prepared in the same
manner as in Example 8 except that pH was set to 4.0 instead of
6.5.
[0173] The preparation included the following steps.
[0174] Hydrochloric acid (0.02 M) (3 ml) was added to dissolve
Bovine Insulin (Sigma) (30 mg), and 2 ml of trisodium citrate (0.2
M), 24 .mu.l of zinc chloride solution (0.12 g/ml), and 0.36 g of
sodium chloride were added. Thereafter, a small amount of HCl (0.1
M) was added to adjust the pH to 4.0. The obtained solution (1 ml)
was transferred to a glass vial, and dried without heating by
centrifugal concentrator under reduced pressure to completely
remove water. To the dried product was added 1 ml of 0.1% w/v
aqueous solution (pH 4.0) of PEG-modified chitosan (manufactured by
Carbomer. Inc.) to give a resuspension as a sample.
[0175] The size of the particles in the obtained suspension was
measured. As a result, the diameter of the particles in the main
resultant product was above 5000 nm (FIG. 15).
Experimental Example 1
Electron Microscope Observation and Elemental Analysis of Small
Particles
[0176] The samples of Examples 10 and 11 were subjected to
observation by FE-TEM (HITACHI, HF-2000, acceleration voltage 200
kV) by dispersion on Cu mesh with a carbon support film and
elemental analysis of the inside of small particles by XMA (Kevex,
Sigma, energy dispersion type).
[0177] FIG. 16 shows a TEM image of small particles of Example 10.
In the small particles of Example 10, surface-coating polymer could
not be observed since the electron density was low. However, egg
albumin particles per se could be observed. Square crystals were
observed in the inside of the particles. As a result of the
elemental analysis of the crystal part by XMA, it was found to be a
crystal of sodium chloride since Na and Cl were contained in large
amounts (FIG. 17). Also, in the TEM image (FIG. 18) of the small
particles of Example 11 in which sodium chloride attached to the
surface of small particles had been removed by dialysis, square
crystals were observed in the inside of the particles and, as a
result of the elemental analysis of the crystal part by XMA, it was
found to be a crystal of sodium chloride since Na and Cl were
contained in large amounts (FIG. 19).
Experimental Example 2
Analysis of Insulin Encapsulation Efficiency in Surface-Coated
Small Particles
[0178] Supernatant (free insulin) was removed from chitosan-coated
insulin particles prepared by the method of Example 4 by
centrifugation (10,000 G, 30 min) to give a particle fraction. 33%
Acetic acid solution was added to the particle fraction to extract
insulin and the insulin concentration was quantified by HPLC. The
encapsulation efficiency was calculated by comparison with the
amount of insulin added by the following calculation formulas:
encapsulation efficiency (%) of added insulin
=100.times.(insulin content mg/ml of prepared surface-coated small
particle suspension)
.times.(total amount ml of prepared surface-coated small particle
suspension)/(total amount mg of added insulin).
[0179] HPLC analysis of the samples was performed under the
following conditions:
[0180] C18 column (Inertsil ODS-2, 5 .mu.m, 250 mm.times.4.6
mm);
[0181] mobile phase A: 0.1% aqueous TFA solution, mobile phase B:
0.1% TFA CH.sub.3CN solution;
[0182] gradient conditions (mobile phase B concentration): at 0
min: 30%, at 10 min: 50%;
[0183] column oven temperature: 40.degree. C., flow rate: 1.0
ml/min, injection volume: 50 .mu.l, detection: UV 275 nm.
[0184] As a result, the encapsulation efficiency of the added
insulin was calculated to be 88%. Therefrom it was confirmed that
the insulin encapsulation efficiency by the above-mentioned
preparation method including dialysis operation was as high as 88%,
and insulin was found to be encapsulated in the particles without a
high degree of loss.
Experimental Example 3
Observation of Changes in Appearance by Preservation at Room
Temperature
[0185] The samples obtained in Example 1 and Example 4 were diluted
with 5 mM MES buffer and visually observed for the initial
appearance and the appearance after standing at room temperature
for one day.
[0186] The results are shown in the following Table 1.
TABLE-US-00001 TABLE 1 observation of changes in appearance after
preservation for one initial day at room temperature Example 1
Colloid completely dissolved suspension Example 4 Colloid Colloid
suspension suspension
[0187] Small particles not coated with a polymer show poor
stability as colloid particles, whereas surface-coated small
particles were stable without dissolution in water even after
long-term preservation. Therefore, it is clear that coating with
polymer can delay dissolution of insulin. From the above results,
use of the surface-coated small particles of the present invention
suggests the possibility of conferring sustained-releaseability to
a natural polymer in vaccine use or DDS use.
Experimental Example 4
Stability Against Enzyme Test of Chitosan-Coated Insulin Small
Particles
[0188] 0.5 ml of the chitosan-coated insulin small particle
suspension (insulin 160 .mu.g/ml, 5 mM MES buffer (pH 6)) of
Example 4 or insulin solution (insulin 160 .mu.g/ml, 5 mM MES
buffer (pH 6)) was mixed with 0.5 ml of model enzyme solution
(.alpha.-chymotrypsin (Sigma Ltd.) dissolved in 5 mM MES buffer (pH
6) at a concentration of 40 .mu.g/ml) and subjected to shaking
reaction at 37.degree. C. for 30 min. After the reaction, cooled
acetic acid (0.5 ml) was added and mixed therewith to quench the
enzyme reaction as well as simultaneously dissolve insulin, and
insulin concentration was quantified by HPLC, based on which
stability of insulin in the chitosan-coated insulin small particles
and stability of insulin in the insulin solution were evaluated. As
a control experiment, an experiment including adding 5 mM MES
buffer (pH 6) instead of the model enzyme solution, and performing
a shaking reaction at 37.degree. C. for 30 min, followed by
quantification of insulin concentration in the same manner as
above, was also performed.
[0189] HPLC analysis of the samples was performed under the
following conditions:
[0190] C18 column (Inertsil ODS-2, 5 .mu.m, 250 mm.times.4.6
mm);
[0191] mobile phase A: 0.1% aqueous TFA solution, mobile phase B:
0.1% TFA CH.sub.3CN solution;
[0192] gradient conditions (mobile phase B concentration): at 0
min: 30%, at 10 min: 50%;
[0193] column oven temperature: 40.degree. C., flow rate: 1.0
ml/min, injection volume: 50 .mu.l, detection: UV 275 nm.
[0194] The results are shown in FIG. 20. The sample of a mixture of
insulin and enzyme showed a decrease by almost 50% in the insulin
content after shaking reaction for 30 min, whereas the sample of a
mixture of surface-coated small particles and enzyme, the sample of
a mixture of surface-coated small particles and buffer, and the
sample of a mixture of insulin solution and buffer showed almost
equivalent insulin contents even after shaking reaction for 30
min.
[0195] From these results, it was confirmed that an insulin
protection effect against enzyme decomposition can be obtained by
coating insulin core particles with a polymer.
Experimental Example 5
Immunity Experiment of Small Particles of Example 10
[0196] 200 .mu.L of small particles of Example 10 (egg albumin
concentration 62.5 .mu.g/ml, pH 6.5) or egg albumin solution (egg
albumin concentration 62.5 .mu.g/ml, pH 6.5) was intradermally
injected to the back of mouse (C57BL/6 lineage). Injection at one
week interval was repeated twice, totaling 3 injections. One week
after final injection, the humoral immunity level and cellular
immunity level of the mouse were evaluated. The humoral immunity
level was evaluated by measuring the amount of antibody against OVA
in the serum based on the absorbance by ELISA method. The cellular
immunity level was evaluated by stimulating the isolated spleen
cells with OVA peptide, counting the spot number of the ELISPOT
method, and measuring the secreted amount of IFN-y.
[0197] The results are shown in the following Table 2.
TABLE-US-00002 TABLE 2 results of mouse immunity experiment of
small particles of Example 10 (N = 2) humoral immunity level
cellular immunity level of serum (absorbance at of spleen cell
(SPOT number 450 nm by ELISA analysis) of ELISPOT analysis) Example
10 0.56, 0.65 (average 0.61) 160, 400 (average 280) OVA 0.23, 0.40
(average 0.32) 77, 46 (average 62) solution
[0198] The small particles of Example 10 were administered to
mouse. As a result, both humoral immunity and cellular immunity
were confirmed to have been strongly induced as compared to
administration of the control solution. Such results indicate that
the small particles of the present invention are useful for vaccine
use.
INDUSTRIAL APPLICABILITY
[0199] Using the pharmaceutical composition of the present
invention, efficient transmucosal or transdermal administration of
a water-soluble drug has been enabled. Such pharmaceutical
composition can be produced by the production method of the present
invention.
[0200] The present invention is based on a patent application No.
2009-231001 filed on Oct. 2, 2009 in Japan, and the contents
thereof are incorporated in full herein.
* * * * *